The cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-1 DNA Hui Zhang, Bin Yang, Roger J. Pomerantz, Chune Zang, Shyamala C. Arunachalam, and Ling Gao A presentation by Alaric Smith

HIV-1 From the Lentivirus category of retroviruses; lentiviri cause immunodeficiency in mammals From the Lentivirus category of retroviruses; lentiviri cause immunodeficiency in mammals Reproduces via reverse transcriptase Reproduces via reverse transcriptase Affects humans, leads to AIDS Affects humans, leads to AIDS

Lentiviral hypermutability In primate lentiviri, G  A transitions common In primate lentiviri, G  A transitions common HIV-1 Vif protein required for viral replication in nonpermissive cells (binds to viral RNA) HIV-1 Vif protein required for viral replication in nonpermissive cells (binds to viral RNA) Is Vif counterdefensive against host nonpermissivity? Is Vif counterdefensive against host nonpermissivity?

CEM15 AKA: Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G, or APOBEC3G AKA: Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G, or APOBEC3G Expressed in nonpermissive cells Expressed in nonpermissive cells Has been shown to inhibit HIV-1 replication Has been shown to inhibit HIV-1 replication Is a cytidine deaminase Is a cytidine deaminase Has been shown to act as a mutagen against E. Coli Has been shown to act as a mutagen against E. Coli

Does CEM15 act on HIV-1 rtDNA? Creation of ∆vif mutant HIV-1 Creation of ∆vif mutant HIV-1 Incubation in permissive (SupT1) and nonpermissive (H9) cells Incubation in permissive (SupT1) and nonpermissive (H9) cells Analysis of newly synthesized viral DNA Analysis of newly synthesized viral DNA

CEM15: An HIV-1 mutagen? ∆vif HIV-1 show significantly higher G  A mutation rate in nonpermissive cells, and when compared to wild-type HIV-1 ∆vif HIV-1 show significantly higher G  A mutation rate in nonpermissive cells, and when compared to wild-type HIV-1 ∆vif viruses passaged in semipermissive (C8166) cells show significantly higher G  A substitution rate than wild-type HIV-1 in C8166 or ∆vif HIV-1 passaged in SupT1 cells ∆vif viruses passaged in semipermissive (C8166) cells show significantly higher G  A substitution rate than wild-type HIV-1 in C8166 or ∆vif HIV-1 passaged in SupT1 cells

How does CEM15 affect G  A substitution? Infect 293T and SupT1 cells (both permissive) with both ∆vif and wild type HIV-1 in presence or absence of CEM15 Infect 293T and SupT1 cells (both permissive) with both ∆vif and wild type HIV-1 in presence or absence of CEM15 After 48h, allow newly synthesized virions to infect C8166 cells, sequence viral DNA After 48h, allow newly synthesized virions to infect C8166 cells, sequence viral DNA Conclusion: G  A hypermutations much more common in ∆vif HIV-1 when CEM15 is present Conclusion: G  A hypermutations much more common in ∆vif HIV-1 when CEM15 is present

How does CEM15 affect wild type HIV-1 over the long term? Transduce CEM15 expression into SupT1 cells Transduce CEM15 expression into SupT1 cells Infect both wild-type and CEM15+ SupT1 cells with wild-type HIV-1 Infect both wild-type and CEM15+ SupT1 cells with wild-type HIV-1 Sequence viral DNA after four passages Sequence viral DNA after four passages Conclusion: Wild type HIV-1 will accumulate G  A hypermutations over time when exposed to CEM15 Conclusion: Wild type HIV-1 will accumulate G  A hypermutations over time when exposed to CEM15 Also, CEM15 does not affect dNTP concentrations Also, CEM15 does not affect dNTP concentrations

Verification of CEM15 cytidine deaminase activity Fuse CEM15 with glutathione S-transferase, purify from E. coli (GST)-CEM15 has significantly higher conversion rate than GST alone, and can be inhibited by tetrahydrouridine

Zinc fingers: An essential domain? In other cytidine deaminases, zinc finger domains are essential for conversion In other cytidine deaminases, zinc finger domains are essential for conversion CEM15 contains two such zinc finger domains CEM15 contains two such zinc finger domains Create several cell lines with mutations in CEM15 zinc finger domains Create several cell lines with mutations in CEM15 zinc finger domains Measure cytidine deaminase activity Measure cytidine deaminase activity

At what level does CEM15 act? Incubate PCR-amplified DNA, in vitro- transcribed RNA, and in vitro reverse-transcribed RNA-DNA duplexes with (GST)-CEM15 Incubate PCR-amplified DNA, in vitro- transcribed RNA, and in vitro reverse-transcribed RNA-DNA duplexes with (GST)-CEM15 Sequence resulting nucleic acids Sequence resulting nucleic acids Result: No discernible mutations were found upon sequencing Result: No discernible mutations were found upon sequencing Conclusion: CEM15 must require other factors to perform cytidine deamination Conclusion: CEM15 must require other factors to perform cytidine deamination

Are CEM15 mutant cells able to defend against ∆vif HIV-1? Incubate ∆vif HIV-1 in CEM15 wild type and mutant 293T cells, then use virions to infect C8166 and HLCD4-CAT cells Incubate ∆vif HIV-1 in CEM15 wild type and mutant 293T cells, then use virions to infect C8166 and HLCD4-CAT cells Analyze viral nucleotide squence Analyze viral nucleotide squence Conclusion: Cells expressing mutant CEM15 effect less G  A substitution than cells expressing wild-type CEM15 in ∆vif HIV-1 Conclusion: Cells expressing mutant CEM15 effect less G  A substitution than cells expressing wild-type CEM15 in ∆vif HIV-1

How exactly does CEM15 work? Observation: Hypermutations preferentially occur in GpA or GpG dinucleotides, or in multiple G groups Observation: Hypermutations preferentially occur in GpA or GpG dinucleotides, or in multiple G groups Proposed mechanism: Direct deamination of dC in newly synthesized viral DNA, causing conversion to dU Proposed mechanism: Direct deamination of dC in newly synthesized viral DNA, causing conversion to dU Could lead to premature stop codons and/or decreased viral protein stability Could lead to premature stop codons and/or decreased viral protein stability Another possibility: Uracil-DNA glycosylase (UDG) may excise uracil from viral DNA, causing break points Another possibility: Uracil-DNA glycosylase (UDG) may excise uracil from viral DNA, causing break points

Future Development What is the biochemical mechanism of CEM15- induced hypermutation? (Is UDG involved?) What is the biochemical mechanism of CEM15- induced hypermutation? (Is UDG involved?) How does Vif neutralize CEM15? How does Vif neutralize CEM15? Does hypermutation ever “backfire?” (i.e., confer added virulence to HIV-1) Does hypermutation ever “backfire?” (i.e., confer added virulence to HIV-1)